Talanta 80 (2009) 1016–1019

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Talanta
journal homepage: www.elsevier.com/locate/talanta

Short communication

Analysis of polyhexamethylene biguanide in multipurpose contact lens solutions夽

Anne D. Lucas ∗ , Edward A. Gordon, Melvin E. Stratmeyer
U.S. Food and Drug Administration, Center for Devices and Radiological Health, OSEL/DB, 10903 New Hampshire Ave, Bldg 64 Room 4011, Silver Spring, MD 20993, USA

a r t i c l e i n f o a b s t r a c t

Article history: The objective of this study was to establish a reasonably simple and reliable method to measure very low
Received 8 June 2009 concentrations of polyhexamethylene biguanide (PHMB) in multipurpose contact lens solutions (MPSs).
Received in revised form 15 July 2009 By using a weak cation exchange solid phase extraction cartridge to extract the PHMB from MPS, fol-
Accepted 16 July 2009
lowed by HPLC analysis using an evaporative light scattering detector, low levels (0.1 ppm) of PHMB were
Available online 23 July 2009
detected. Application of this method to a series of off-the-shelf MPS with PHMB as the active ingredient
demonstrated these solutions contain 1 ppm. The contact lens solution with hydrogen peroxide as the
Keywords:
active ingredient gave no peak where the PHMB peak eluted. The Polyquad® contact lens solution gen-
Contact lens
Polyhexamethylene biguanide
erated a peak close to the retention time of PHMB. Recovery of PHMB from fortified hydrogen peroxide
Liquid chromatography contact lens solution was good at 0.25 ppm and above; 105% with a RSD of 17% or less. The repeatability of
the HPLC system ranged from 4 to 11% RSD; the reproducibility of the entire method was less than 17.5%
RSD. Storage and stability studies indicated that storage of MPS with PHMB for chemical analysis are not
temperature dependent, but are affected by the composition of the container in which the contact lens
solution is stored.
Published by Elsevier B.V.

1. Introduction tion. A capillary electrophoresis (CE) method [10] for determination

PHMB1 (polyhexamethylene biguanide) is a chemical germicide
used in a variety of applications: as a preservative in wet wipe, to
prevent microbial contamination in wound irrigation and sterile
dressings, and to disinfect medical/dental utensils and trays. PHMB
is also used as an active ingredient for recreational water treatment,
in cosmetics, personal care products, fabric softeners, contact lens
solutions, and hand washes [1]. This widely used biocide has been
reviewed by EPA and noted, with the exception of occupational han-
dlers, as having very low aggregate risk of adverse health effects to
the public or environment [2].
This compound is a challenge to quantitate at low levels because
it is a mixture of polymers with no distinctive chromophores [3] and
is a polyelectrolyte with a mix of end groups [4]. Dye-binding assays
[5–7] have limits of detection around 5 ppm and greater; the con-
centration of PHMB in some contact lens solutions is 1 ppm. High
pressure liquid chromatographic (HPLC) using photodiode array
detectors also are not sensitive enough because UV absorption of
PHMB is too weak [7–9]. The mix of different end groups of the
PHMB polymer prevents the possibility of quantitative derivitiza-
夽 The mention of commercial products, their sources, or their use in connection
with material reported herein is not to be construed as either an actual or implied
endorsement of such products by the Department of Health and Human Services.
∗ Corresponding author. Tel.: +1 301 796 0283; fax: +1 301 796 9826.
E-mail address: anne.lucas@fda.hhs.gov (A.D. Lucas).
1 2
Polyhexamethylene biguanide.
of PHMB in eye drops was described with a limit of detection of
4 ppm. Other means of PHMB analysis include titrimetric methods
[11] that do not have adequate levels of sensitivity or potentiometric
titration methods that involve custom synthesis [12]. There appears
to be no established method for measuring PHMB in contact lens
solution or at sub-ppm levels.
In this paper, a method to reliably measure PHMB at sub-ppm
levels in MPS2 (multipurpose contact lens solution) is detailed. This
was accomplished by developing a solid phase extraction method
followed by HPLC analysis using an evaporative light scattering
detector.
2. Materials and methods
2.1. Reagents and solutions
Several commercially available brands of MPS were purchased
from local stores; six different brands contained PHMB as the active
ingredient, two contained hydrogen peroxide, and the last one
contained Polyquad® as the chemical disinfectant. Methanol and
acetonitrile were obtained from Fisher Scientific (Waltham, MA,
USA), and triethylamine (TEA), trifluoroacetic acid (TFA), and formic
acid (FA) were from Sigma–Aldrich (St. Louis, MO, USA). Deionized
water was produced with a Barnstead NANOpure Diamond water
Multipurpose contact lens solution.

0039-9140/$ – see front matter. Published by Elsevier B.V.

doi:10.1016/j.talanta.2009.07.031
 A.D. Lucas et al. / Talanta 80 (2009) 1016–1019
purification unit. The PHMB, trade name Cosmocil CQ (a 20% solu-
tion in water), was graciously donated by Arch Chemical Company
(Norwalk, CT, USA).
2.2. HPLC method
Samples were analyzed using a Waters HPLC system, equipped
with 515 pumps, a 717 plus autoinjector, a 996 photodiode array
detector and an evaporative light scattering detector (PL-ELS2100,
Polymer Laboratories) with Waters Empower chromatographic
software. Settings for the evaporative light scattering detector
were: evaporator 111 ◦ C, nebulizer 80 ◦ C, zero-grade nitrogen at 1.6
SLM (standard liters per minute). The photodiode array detector
was set at 254 nm, but was not used for quantitation. Separation was
performed on a Supelco SupelcosilTM LC-8 column (5 cm × 4.6 mm,
5 ␮m). The mobile phase component was 100% deionized water
held isocratically for 4 min, then an immediate change to 50:50
water: a solution composed of acetonitrile (76%), water (9.5%), tri-
ethylamine (5%), and formic acid (9.5%), pH adjusted to 3.1 with
formic acid for the next 2 min, then a linear gradient back to 100%
water at 10 min. The flow rate was 2 ml/min and the injection vol-
ume was 0.25 ml.
2.3. Extraction of contact lens solutions
Extraction and concentration of PHMB from MPS were per-
formed with weak cation exchange solid phase extraction cartridge
from Waters (WCX Oasis 1 cc, 30 mg). Cartridges were first pre-
washed with two column volumes of 50:50 acetonitrile:distilled
deionized water with 0.25% TFA, then two column volumes of
acetonitrile, followed by two column volumes of distilled deion-
ized water. Five ml of sample were then loaded onto the cartridge
followed by washing with two column volumes of water, then
with 0.5 ml of 50:50 acetonitrile:water with 0.02% TFA. PHMB
was eluted with 0.5 ml of 50:50 water:acetonitrile with 0.25% TFA
directly into 8 mm × 40 mm HPLC vials. Injection volume for the
HPLC was usually 0.25 ml. HPLC vials were pre-washed with 50:50
water:acetonitrile with 0.25% TFA, then acetonitrile and left to air
dry prior to use.
2.4. Storage stability studies
MPS brand A was used to examine the storage and stability
of PHMB at three different temperatures (25 ◦ C, 4 ◦ C, and −20 ◦ C).
Using aseptic technique, approximately 20 ml of the MPS was
placed in each of 12 separate 50 ml polypropylene test tubes. Four
of these test tubes were left at room temperature (25 ◦ C); four
were placed in the refrigerator (4 ◦ C), and four in a standard freezer
(−20 ◦ C). Over time, one test tube from each different temperature
locale was removed and triplicate aliquots of 5 ml were extracted as
previously described. In addition, the stability of PHMB in glass vials
compared to polypropylene test tubes was examined by aseptically
placing 20 ml of MPS brand A in glass or polypropylene containers
and leaving them overnight at room temperature. The following
day, triplicate extracts of the MPS brand A stored in glass vials was
compared to triplicate extracts stored in polypropylene test tubes.
3. Results and discussion
3.1. Evaluation of dye binding methods
Initially, the colorimetric methods were explored because of the
potential for high throughput and low cost. The method described
by Rowhani and Lagalante [7] was tested first. However, the
absorbance from a MPS with PHMB was roughly 100 times higher
than the PHMB standard concentration. Other compounds in the
1017
MPS were probably exerting a large positive influence in this assay.
The second colorimetric assay evaluated was detailed by Rosen-
thal et al. [5]. Again, the MPS overestimated the amount of PHMB,
but only by a factor of four. The third colorimetric assay [6] over-
estimated the PHMB concentration in MPS by a factor of five. In
addition, none of these assays were very sensitive; all the colori-
metric methods had limits of detection around 5 ppm or greater.
Because MPS labels state a concentration of 1 ppm of PHMB, a more
sensitive and selective method was needed.
3.2. Solid phase extraction
A series of solid phase extraction cartridges were evaluated in
sample extraction. Although PHMB is a cation, an anion exchange
column and a lipophilic column were examined to see if they would
trap interfering substances while permitting PHMB to pass through.
However, significant interfering substances eluted with the PHMB
from both the lipophilic and strong anion exchange columns. The
weak and strong cation exchange columns were used to trap the
PHMB while allowing most interfering substances to elute. Because
it was possible to elute PHMB from the weak cation exchange col-
umn using less caustic conditions, this was the cartridge chosen.
Coupling the solid phase extraction method to the colorimetric
assays did not work as the strong acid needed to elute the PHMB
occupied the binding sites for the dyes.
3.3. Detector performance
Although the reproducibility of the evaporative light scattering
detector (ELSD) has been noted to be worse than UV [13] and the use
of a gradient increases the variability of the analyte signal [14], this
is a more sensitive detector for compounds without chromophores.
The light scattering by the solute after removal of the eluent is mea-
sured [13]; detection is dependent on the amount of solute. Fig. 1
illustrates the difference between using an ELS detector (Fig. 1A)
and a UV detector (Fig. 1B) in detecting PHMB extracted from con-
tact lens solution brand A. The Refractive Index detector is useful
for detecting compounds that do not absorb UV, it is not useful for
gradient elution [15]. Attempts to generate a useful ion for mass
spectroscopy with electrospray or atmospheric pressure ionization
were not fruitful.
3.4. Recovery
MPS contain salts, surfactants, and other additives, as well as the
active biocide; although not all brands are identical chemically or in
quantity. Because the hydrogen peroxide contact lens solution gave
no peak near the retention time of PHMB, it was used in the recovery
studies as the best available approximation of a blank MPS. Recov-
ery of hydrogen peroxide MPS fortified with 0, 0.125, 0.25, 0.5, and
1.0 ppm PHMB are depicted in Fig. 2. The recovery was determined
from triplicate experiments each with triplicate samples. Recover-
ies were good, 107%, at the 0.25 ppm level and above (RSD 17% or
less), but the error at the 0.125 ppm level was quite large (RSD 66%).
The bias towards high recovery at low levels is likely due to inter-
gration errors because of the steep gradient used and the increasing
background and error of the detector over time (Section 3.6 Table 1).
3.5. Evaluation of commercially available contact lens solutions
Six different MPSs with PHMB as the active ingredient were pur-
chased at local stores and analyzed. Fig. 3 details the PHMB levels
found in these six different MPS with PHMB; the data represent
triplicate experiments with each contact lens solution run in tripli-
cate. The PHMB concentration in all the MPS was 1 ppm with RSD of
17.5% or less; this supported the utility of the method. The contact
1018 A.D. Lucas et al. / Talanta 80 (2009) 1016–1019

Fig. 1. The difference in the sensitivity of the ELSD detector (A) and the UV detector (B) for PHMB analysis of contact lens solution brand A.

lens solution with hydrogen peroxide as an active ingredient gen- peak area was three times larger than that of the PHMB. It might
erated no peak in the PHMB area. The MPS containing Polyquad® be possible to tease the Polyquad® peak away from the PHMB by
generated a large peak that appeared 0.05 min ahead of the PHMB using a gentler gradient. The purpose of using an immediate change
peak (data not shown). It can be possible to confuse this peak with in pH and mobile phase composition in this study was to generate
PHMB based on the close retention times; however the Polyquad® one peak for PHMB [16]. Other investigators reported the need to
integrate multiple peaks to quantitate PHMB [7]. The purpose of this
Table 1 study was to develop a sensitive reliable method for PHMB in con-
The repeatability of the system as a function of peak area for a 10 ppm PHMB tact lens solutions. The baseline separation of the Polyquad® from
standard. the PHMB peak was not considered to be necessary as, at present,
Peak area
both chemical disinfectants are not found together in commercially
available MPS.
Number Average Std cv%

6 1,311,365 135,376.7 10.3

5 1,423,358 159,430.7 11.2 3.6. HPLC system repeatability
10 3,171,998 145,270.5 4.6
8 4,170,078 318,143.5 7.6 The error of the system was evaluated by making a large volume
8 4,412,432 172,020 3.9
of a 10 ppm PHMB in the elution solution (50:50 water:acetonitrile
 A.D. Lucas et al. / Talanta 80 (2009) 1016–1019
Fig. 2. Recovery of PHMB fortified with 0, 0.125, 0.25, 0.5, and 1.0 ppm into hydrogen
peroxide contact lens solution. Recoveries were good, 107%, at the 0.25 ppm level and
above (RSD 17% or less), but the error at the 0.125 ppm level was quite large (RSD
66%).
Fig. 3. The average concentration of PHMB in six different commercially available
MPSs.
Fig. 4. The effect of temperature on the storage of PHMB, MPS brand A was used for
this study.
with 0.25% TFA) and placing it into a series of HPLC vials. Single
injections were made for each set of standards; the number of the
10 ppm standards prepared varied over time (Table 1). The variabil-
ity of the peak area for the same concentration of PHMB was due
to the need to ‘steam clean’ the ELSD every 40 h of use. Standards
were run over a period of two months at various time points before
and after the detector cleaning. Just after steam cleaning the detec-
tor, the peak area was largest and peak shape was sharpest. Table 1
summarizes the data; that increasing peak area shows decreasing
error expressed as RSD%.
1019
3.7. Storage and stability studies
Storage of MPS brand A was evaluated at room temperature, 4 ◦ C,
or −20 ◦ C for up to a four week period. No significant difference in
total PHMB concentration was seen. Fig. 4 illustrates the temper-
ature stability of MPS brand A; the RSD for this data set was less
than 10%. When MPS brand A was placed overnight at room tem-
perature in a glass container and its peak area was compared to the
same MPS stored in a polypropylene container at the same tem-
perature, a 50% decrease in signal was observed (data not shown).
This is likely due to the negative charge of the glass [17] binding the
cation PHMB.
4. Conclusion
A reasonably simple method for the determination of PHMB in
MPS was developed. Recovery from fortified solutions and com-
mercially available MPS containing PHMB were around 100% at the
relevant 1 ppm PHMB level, with a limit of detection at around
0.2 ppm. All commercially available MPS containing PHMB that
were tested had concentrations around the labeled 1 ppm level.
The storage stability studies conducted demonstrated that the tem-
perature (25 ◦ C to −20 ◦ C) did not negatively impact the chemical
analysis of PHMB from contact lens solutions over 28 days. In com-
paring glass and polypropylene containers for contact lens solution
storage, polypropylene is superior to glass for maintaining the con-
centration of PHMB in MPSs. However, with out analyte-free MPS,
the true accuracy cannot be unequivocally established. Still, the cir-
cumstantial evidence would support this method as a reasonable
means for determining PHMB in MPS.
Acknowledgements
The authors wish to thank Mr Peart, Arch Chemical Co, for donat-
ing PHMB, Gail Matson for her editorial expertise, Ann Koustenis for
laboratory support, and Dr. Seungil Cho and Dr. Victoria Hitchins for
the technical review.
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